Intermediate filaments comprise an integral part of the neuronal cytoskeleton. However, little is known about their function, and there remains some uncertainty about their precise subcellular localization. We examined the timing of expression and distribution of α-internexin, neurofilament triplet proteins and peripherin using immunocytochemistry in cultured hippocampal neurons. α-Internexin immunostaining was present in all neurons at all developmental stages. Immunostaining appeared as long filaments in axons and short fragments in dendrites which extended into dendritic spines. The presence of α-internexin in dendritic spines was confirmed in situ by electron microscopy of rat hippocampal tissue sections and suggests that this intermediate filament may serve as a link between cytoskeletal elements in dendritic shafts and spines. In culture, immunostaining using antibodies against individual triplet protein subunits indicated that light (NF-L) and middle (NF-M) subunits were first expressed in cells shortly after the initiation of axonal outgrowth. Expression of the heavy (NF-H) subunit occurred a few days later. Although timing and localization of expression did not correlate with the initiation of axonal or dendritic processes, it was coincident with periods of rapid outgrowth. Triplet proteins were more abundant in axons and appeared to be incorporated into lengthier filaments than in dendrites. Highly phosphorylated NFH/M immunoreactivity was polarized to axons after 6 days in culture. The distribution of one NF-H epitope was restricted to GABAergic neurons in mature cultures, suggesting a cell-type specific modification. Peripherin was not detectable at any time in hippocampal cultures. Our results show that intermediate filaments are integral components of the neuronal cytoskeleton of cultured hippocampal neurons throughout development. Furthermore, the localization of α-internexin suggests that it may be involved in the formation or maintenance of dendritic spines.